How do adamantane drugs block M2?

Vaccination plays such an important role in our seasonal influenza strategy in part because we don't have many medicines that can be brought to bear on the disease. The neuraminidase inhibitors (specifically Tamiflu) are widely stockpiled, and continue to work for now, but the specter of resistance is already lurking. If these drugs are too widely or too improperly used, there is a good chance that resistance mutations will eventually render these drugs ineffective. Universal drug resistance may already be the fate of the drugs amantadine and rimantadine, built on an adamantane backbone (1). The adamantane drugs inhibit the M2 proton channel from influenza A, a tiny tetrameric protein that equalizes pH between the virus and the endosome of the cell that has swallowed it. This process releases the virus contents so that they can do their damage to the cell, so these medicines can significantly retard the infection process. Or rather, they could, if so many influenza strains didn't harbor the S31N mutation that almost completely nullifies their effect. If we are to develop new drugs to attack the M2 channel, it would be helpful to know how this mutation causes drug resistance. Over the past few years a great deal of structural evidence has accumulated showing how adamantane drugs work on the older, non-resistant channels. The problem is that the evidence supports two different models of M2 inhibition, and so far it has proven difficult to determine which of them is probably correct.

How the question arose

The controversy is the result of two structures published in Nature early in 2008 (2,3). The first of these is a crystal structure of a tetramer of peptides encompassing the transmembrane (TM) region of the M2 channel reported by the DeGrado group at UPenn, which you can see at right (explore this structure at the PDB, noting that the numbering is off by 21). In the detergent used for crystallization, the peptides form a tetramer with a roughly conical pore, which amantadine (purple in these models) physically occludes, giving rise to the pore-blocking model (PBM). This model is consistent with previous results indicating that a single amantadine molecule is sufficient to inhibit the proton channel. In addition, in this model the drug binding site is adjacent to S31 (blue side chain), which is what we'd expect given that an S31N mutation is responsible for most amantadine resistance. The authors propose, given the position of the S31 side chain, that the mutant asparagines form a hydrogen-bonded network that is too constricted for amantadine to bind. Click on the picture for a larger view.

An alternative model was proposed by Schnell and Chou from Harvard University (3). They produced an NMR structure (left) of a 42 amino-acid peptide from M2 encompassing the TM region and an additional C-terminal helix (explore this structure at the PDB). In their structure, taken at pH 7.5 in detergent micelles, the tetramer forms a roughly cylindrical pore that is blocked by the side chains of the known gating residues W41 and H37 (light green in these models). Their structure shows rimantadine bound at four sites near the base of the helix but not in the pore. Using pH-dependent conformational exchange experiments, Schnell and Chou showed that a decrease in pH caused rapid structural changes in the channel, motions that rimantadine slowed. On the basis of this evidence, they proposed a mechanism in which protonation of the gating histidines destabilizes the packing of the TM helices and allows the conductance of protons. Rimantadine blocks the channel by stabilizing the helices, thus this is a dynamic quenching model (DQM). The position of S31 in this model is also somewhat different than the crystal structure, although these models were made at different pH conditions and so this may represent a difference between the closed and open states of the channel.

The distinction here is important. If Stouffer et al. are correct, then drug development should abandon the adamantane backbone altogether and start with a set of significantly different leads to address the resistance problem. The PBM implies that any molecule large enough to occlude the pore will be too large to fit in there following the S31N mutation that induces amantadine resistance. If the DQM is correct, however, then it is conceivable that further refinements to the adamantane base, or similar molecules, could improve affinity enough to overwhelm the mutational effect.

Unfortunately, neither result is unimpeachable. Although it agrees with a great deal of experimental evidence, the low resolution of the crystal structure means that the electron density called amantadine cannot be assigned unambiguously. It is also curious that a hydrophobic molecule like amantadine would bind tightly in the hydrophilic pore. In addition, the crystal form with amantadine bound contains a mutation, G34A (black side chain), which is near the drug binding site and could conceivably have altered the binding specificity of the protein.

The NMR structure has the advantage that it directly includes distance information in the form of NOEs. However, the authors used 40 mM rimantadine to obtain these results, meaning that there were as many rimantadine molecules in the solution as phosphate buffer molecules. Under these conditions, it is possible that the drug bound to a secondary, low-affinity site. Even if this is what happened, it is strange that the rimantadine never bound to the high-affinity site indicated by the crystal structure.

Both experiments use significantly truncated constructs and highly artificial systems to mimic a membrane environment. The structure of any membrane protein depends in often unexpected ways on the composition of the lipid bilayer in which it is embedded and on the structure of that bilayer. The intense curvature of the micelles may have distorted the structure in the NMR experiment, and possibly inappropriate lipids may have had effects on both structures. We know these considerations are relevant for this system, because Schnell and Chou report that the construct used for the crystal structure would not form stable tetramers in the micelle system. Also, as Chris Miller notes in his commentary on these papers (4), there were questions about both constructs with respect to their proton conductivity. Lacking significant stretches of the protein and placed in these environments, it is possible that both structures deviate from in vivo reality in significant ways.

Because the conditions diverge so much, it is difficult to weigh the mechanisms based on these structures alone. The binding site identified by Schnell and Chou is only at the very end of the construct used by Stouffer et al.. In addition, the inhibited crystal structure comes from a low-pH condition while the NMR structure exclusively represents a high-pH condition. Given these differences in conditions, it is not impossible that both models, in whole or in part, are correct. We must turn to additional experiments and alternative evidence to choose between them, specifically data on the stoichiometry of binding and the effects of mutations.

Binding stoichiometry

The crystal structure shows a single binding site for the drug, while the NMR structure implies four, and this is at odds with existing results that indicate that a single molecule of drug is sufficient to inhibit a single channel. Given the homotetrameric nature of the M2 channel, it is in principle not possible for the NMR experiment to distinguish between a single rimantadine binding event and four. That is, the NMR experiment cannot tell us whether the rimantadine-M2 inhibition occurs with a single binding event or requires four drug molecules to bind. Therefore, to argue that DQM is inconsistent with 1:1 stoichiometry overstates the case somewhat.

It may also be somewhat overstating the case to say that there is only one amantadine binding site on M2. Washing amantadine out of your buffer does not reverse inhibition, in part because of slow kinetics of leaving the binding site and in part because these drugs, being very greasy, preferentially partition into the lipid membranes and are therefore not readily removed from a system when its aqueous phase is replaced. It is difficult to measure a binding constant for the drugs because the equilibria under consideration will be quite complex. The studies often cited on the 1:1 stoichiometry (5,6) use structural and kinetic evidence to get at this question.

Czabotar et al. (5) measured tryptophan fluorescence in M2 as a function of pH and rimantadine concentration. They found that fluorescence from W41 was quenched by decreased pH, but recovered when 1 equivalent rimantadine per tetramer was added. This result implies that structural or dynamic changes caused by histidine protonation are reversed by rimantadine inhibition, but this is so general that it cannot be taken to support either the PBM or DQM.

Wang et al. (6) measured the reduction of surface currents in X. laevis oocytes after addition of various concentrations of amantadine. From these results they are able to construct a Hill plot with a coefficient of 1, showing that binding of amantadine is not cooperative. In further results, Wang et al. find that amantadine inhibits M2 channels slightly better at high pH (when the pore is closed) than at low pH, and that amantadine inhibits proton conductance in either direction (rather than favoring one). Both these outcomes are unexpected for PBM, but can be easily explained by DQM. However, the differences in the binding constants are relatively minor and the linearity of the current-voltage relationship may result from some other idiosyncratic feature of the M2 channel, so these results are not unequivocal.

Neither experiment refutes DQM because they do not measure the number of binding sites, but rather the number of efficacious binding sites. If there are four binding sites, but 95% or more of the inhibitory or structural effect is caused by the first drug molecule bound, then these experiments would be unable to distinguish DQM from PBM. Overall, the evidence on the question of binding stoichiometry does not eliminate the possibility of four binding sites existing, but it does place a requirement on DQM that the inhibitory effect of amantadine on the tetramer result from a single binding event. Because the proposed DQM binding site for rimantidine lies between monomers and is linked to the gating tryptophan, this is not unbelievable. Other evidence from these experiments is equivocal, but can be seen as somewhat more problematic for PBM than DQM.

Functional effects of mutations

A serious problem for DQM is that the mutations known to give M2 resistance to adamantane drugs are all located near the PBM binding site. In particular, S31 is adjacent to the drug in the crystal structure and quite distant in the NMR structure. As Miller notes in his commentary, mutational studies are substantially more difficult to interpret than is typically suggested, so this isn't absolutely probative. In general, however, one predicts mutations to have short-range rather than long-range effects, so at least some resistance mutations ought to evolve at the binding site. However, many of the residues surrounding the DQM site are almost absolutely conserved, presumably because they are essential to the function of the channel. As a result, it would be very difficult to interpret any studies on point mutants in this area. What would be ideal, however, would be to find a set of mutations that produced a functional protein and abrogated amantadine inhibition.

This is the basis for an interesting experiment conducted by the lab of Robert Lamb and reported last year in PNAS (7). In this case, the authors took advantage of the fact that the M2 protein from influenza B virus is not sensitive to adamantane drugs. They constructed a chimeric protein containing about a dozen residues from influenza A M2 — specifically, the dozen or so residues surrounding the PBM site. If PBM is correct, then we would expect that these residues, which define that site, would impart amantadine susceptibility to the influenza B channel. This is what happens, sort of. For your benefit, I have shamelessly stolen their figure (right), but you can check out this paper yourself because it is open access. In this assay, again involving X. laevis oocytes, the hybrid channel is sensitive to amantadine (bottom trace), but only half as sensitive as the wild-type influenza A channel (second from top). This result suggests that there is important context conferring susceptibility outside the PBM site. However, this could be something as simple as helix orientation, so the result does not necessarily imply that there is an external binding site.

Additionally, the authors made point mutations at residues (L38, D44, and R45) that were presumed to be important in the DQM mechanism or have long-range effects on amantadine binding. None of these mutations appeared to affect amantadine resistance. In contrast, experiments in liposomes reported by the Chou group this May showed that a D44A mutation prevented rimantidine from having an effect (8). This conflict in results is difficult to reconcile, but may result from the different constructs used (the Chou group used a truncated form of M2 while the Lamb group used the full-length protein) or from changes in ion specificity caused by the D44A mutation. It might be of value to repeat these experiments with the alternative construct: truncated in oocytes, full-length in liposomes. Because the D44A mutant does not appear to conduct protons as efficiently as WT, the proposition that the function of this mutant is too deranged to provide trustworthy information should also be considered.

Additional experiments in the Chou paper are meant to address the relationship between the DQM site and the mutations at the PBM site. They show that the S31N mutation prevents rimantidine binding to the remote site, and also that this mutation makes the protein generally more dynamic. From this evidence they propose that this mutation, at least, disrupts amantadine binding by destabilizing the helical packing of the channel and thus interfering with the organization of the lipid-facing pocket.

They also examine an S31A mutation and find that it is not rimantadine-resistant or destabilizing to the packing. This supports their dynamic model in a limited way, because it demonstrates that only certain mutations at the S31 site will generate resistance. It does not cast any doubt on PBM, however, because in that model resistance in the S31N mutant is explained by the idea that its side chain will partially obstruct the pore so that rimantadine will not fit. I do not think it was ever proposed that specific contacts between S31 and the drug stabilize the binding; in fact, the general absence of such contacts strikes me as a concern about PBM.

Chou et al. also examine the effect of rimantadine on the shorter construct used for the crystal studies. They find that the inhibition of this construct is substantially weaker. However, it also conducts protons at a much slower rate in this assay, suggesting that there may be additional serious problems with the function of this construct. It may be that it simply is not appropriate to use this construct for studies in solution or living membranes. That doesn't necessarily imply that this peptide will give incorrect information in the stabilizing environment of a crystal.

What do we know, and what do we need?

Very little of this evidence unequivocally prefers one model to the other. We know that a single adamantane molecule is sufficient to inhibit M2, and while this is most obviously compatible with PBM it need not be inconsistent with DQM. It is also apparent that various constructs of the M2 channel retain adamantane susceptibility after ablation of the DQM site, either by truncation, mutation, or the construction of a chimeric protein. In all assays, however, the adamantane drugs lose a considerable amount of inhibitory power, so these results are not entirely consistent with PBM either. And, at least in the Chou lab's assays, interference with the DQM site also reduces adamantane susceptibility and deranges function. Moreover, the NMR data from the Chou lab shows that mutations at PBM site have a long range effect on the DQM site, which mitigates the probative power of the S31N mutation.

How do we address this question? One important step would be to start comparing like to like. We are considering evidence from a plethora of constructs and conditions, and the evidence in conflict is often collected in very divergent experiments. Ideally we would like to have structures of the wild-type channel at low and high pH in a lipid environment that closely mimics the composition and curvature of a mature influenza virion. As this is unlikely in the near term, we must hope for NMR and crystal structures that at least use the same construct, minimally mutated, under similar conditions. NMR studies at low pH would be of value in assessing whether these studies in fact contradict one another. Additionally, it would be useful to make adamantane derivatives labeled with a free radical or other paramagnetic label; this would presumably allow the identification of a binding site at lower drug concentrations in an NMR experiment. Labeling the drug with a metal might also allow its identification in a crystal structure without any need to push the resolution significantly higher. Finally, actual structures of the S31N mutant, positively identifying the disposition of this side chain, would be of great value in judging the question.

Structural experiments can take a great deal of time and careful tuning, a requirement exacerbated by the often-fickle behavior of membrane proteins. As such, additional mutational studies could prove useful. Inverting the chimera experiment of Jing et al. to create a chimeric protein with the upper channel from influenza B and the lower channel from influenza A may be a helpful supplement to the existing experiments. If the C-terminal portion of the channel makes a contribution to adamantane inhibition this chimera will also be rimantidine-sensitive. In addition, new mutations at S31 could help distinguish the possibilities. The PBM supposes that the N31 side chains stick into the pore and form hydrogen bonds, while the DQM supposes that they stick into the interface of the TM helices and destabilize them. An S31L mutant should disrupt the helical packing but not form hydrogen bonds or extend the L31 side chains into the pore. If functional, such a mutant ought to be rimantidine resistant if the DQM is correct, but not if PBM is correct. Assuming the geometry of the longer side chain is wrong for formation of a hydrogen bonding network, an S31Q mutation might be useful as well. Similarly, mutations that increase the size of the L40, I42, or L43 side chains could prevent adamantane binding to the DQM site without degrading the channel's transport capabilities; the drug sensitivity of such a mutant would be a powerful argument either way. Any experiments of this kind would likely be easier to perform than to interpret, but could provide valuable insight. Obviously, it would also be important to establish that each mutant was competent at transporting protons.

The unspoken assumption of the debate so far is that these mechanisms are mutually exclusive, but there is no particular reason to believe this must be so. The structural experiments definitively show that binding to both sites is at least possible — even if one clings tenaciously to the idea that the density observed in the crystal is not in fact amantadine, that structure at least shows that the PBM site is capable of accommodating the drug. It might therefore be plausible that adamantane drugs inhibit M2 using both mechanisms simultaneously, or that DQM predominates at high pH and PBM at low pH. Redundancy in inhibitory mechanisms may explain the curious features of amantadine inhibition noted by Wang et al., and the inability of experiments specific to a single site to completely account for adamantane inhibition. In addition, the fact that S31N interferes with both mechanisms may explain why it is the primary resistance mutation.

An experiment with the alternate chimera mentioned above could test this possibility. In addition, if the mechanisms switch off in a pH-dependent fashion, then this should be testable with the hybrids: specifically, the A/B M2 used by Jing et al. should have lower susceptibility to adamantane drugs at high pH than at low pH. Similarly, the B/A M2 chimeric protein, if inhibited by amantadine, would be more resistant at low pH.

Doubtless these suggestions are nothing new to the members of the labs working on this perhaps unexpectedly hairy question. Membrane protein structure and function is one of the most difficult experimental subjects in biochemistry, and constitutes a critically important frontier in scientific efforts to improve human health. It is infinitely easier to propose most of these experiments than it is to perform them, and I would be remiss if I did not temper the persistently critical tone of this post with some praise for the efforts of all the scientists involved in this research, and for their commitment to getting the right answer. These papers represent years of work by incredibly talented people using some of mankind's most advanced scientific techniques. The lack of clarity on the question of adamantane drugs binding to M2, even in the face of this amazing effort, is a testament to the enormous difficulty of researching these critical systems.

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1. Deyde, V., Xu, X., Bright, R., Shaw, M., Smith, C., Zhang, Y., Shu, Y., Gubareva, L., Cox, N., & Klimov, A. (2007). Surveillance of Resistance to Adamantanes among Influenza A(H3N2) and A(H1N1) Viruses Isolated Worldwide The Journal of Infectious Diseases, 196 (2), 249-257 DOI: 10.1086/518936 OPEN ACCESS

2. Stouffer, A., Acharya, R., Salom, D., Levine, A., Di Costanzo, L., Soto, C., Tereshko, V., Nanda, V., Stayrook, S., & DeGrado, W. (2008). Structural basis for the function and inhibition of an influenza virus proton channel Nature, 451 (7178), 596-599 DOI: 10.1038/nature06528

3. Schnell, J., & Chou, J. (2008). Structure and mechanism of the M2 proton channel of influenza A virus Nature, 451 (7178), 591-595 DOI: 10.1038/nature06531

4. Miller, C. (2008). Ion channels: Coughing up flu's proton channels Nature, 451 (7178), 532-533 DOI: 10.1038/451532a

5. Czabotar, P., Martin, S.R., & Hay, A.J. (2004). Studies of structural changes in the M2 proton channel of influenza A virus by tryptophan fluorescence Virus Research, 99 (1), 57-61 DOI: 10.1016/j.virusres.2003.10.004

6. Wang, C., Takeuchi, K., Pinto, L.H., & Lamb, R. (1993) Ion Channel Activity of the Influenza A Virus M2 Protein: Characterization of the Amantadine Block J. Virol. 67 (9) 5585-5594 Available free from PubMed Central

7. Jing, X., Ma, C., Ohigashi, Y., Oliveira, F., Jardetzky, T., Pinto, L., & Lamb, R. (2008). Functional studies indicate amantadine binds to the pore of the influenza A virus M2 proton-selective ion channel Proceedings of the National Academy of Sciences of the United States of America, 105 (31), 10967-10972 DOI: 10.1073/pnas.0804958105 OPEN ACCESS

8. Pielak, R., Schnell, J., & Chou, J. (2009). Mechanism of drug inhibition and drug resistance of influenza A M2 channel Proceedings of the National Academy of Sciences of the United States of America, 106 (18), 7379-7384 DOI: 10.1073/pnas.0902548106

The Scientific Activist and Discovering Biology in a Digital World also have interesting posts on this subject.

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Here there be dragonflies

Because we constantly interact with each other, we often forget that human beings are absurd, freakishly huge creatures. Of course you and I and any four-year-old can reel off the names of a dozen or so animals that are even larger, but that's misleading. The earth is inhabited by trillions upon trillions of animals, and almost none of them grow larger than our own hands. These creatures inhabit a world we can barely recognize, the objects we find familiar made exotic by the transition of scale, and yet their lives have drama and tension more than equal to our own. Deadly Creatures successfully sets up this atypical viewpoint, but falters because its narrative and gameplay adhere strictly to a human perspective.

You might expect a game about a tarantula and a scorpion to look like a National Geographic special, but Deadly Creatures eschews the crisp brightness of magazine shots in favor of a muted palette more reminiscent of the Metroid Prime series. The comparison seems even more apt in light of the almost alien character the desert landscapes take on, not just because of the changes in scale but also because of the variability of orientation. Deadly Creatures doesn't just put you in a scaled-up version of a pickup truck; it forces you to explore that space using every conceivable surface as a floor. The game also seems to share Metroid Prime's obsession with ruins, though here they are scaled down. The leftovers of human civilization — a cast-off boot, a broken lawn gnome — have become the homes of your dangerous enemies.

The comparisons go deeper than the aesthetics. Deadly Creatures comes across as a game of exploration, using the tiny grubs like Metroid Prime's copious power-ups to encourage you to see everything it has to offer. While it lacks something in variety, this approach fits relatively well with the game's fiction. Additionally, Deadly Creatures loves to return you to previously-visited areas with new abilities that allow you to take alternate routes across and out of them.

Where this comparison begins to break down is in the way the game tries to guide the player towards goals. Metroid Prime makes the player a dynamic actor in a relatively static world and lets him go where he will, his access limited only by the abilities he has acquired. While the player is not exactly free to choose his objectives, the motif of restricted access makes them feel like an organic part of the game's intrinsic fiction. Deadly Creatures artlessly blocks off particular exits with clouds of flies in an effort to push the player along in a set story arc.

The story itself is the most obvious example of the game's key failing, because it concerns human beings operating with specifically human motivations. Billy Bob Thornton and Dennis Hopper do a wonderful job with voice roles that simply don't fit in this game. What does buried treasure have to do with a tarantula and a scorpion? The game never bothers to make this clear, but it gives the player objectives that force him down a path towards confrontation. The goals may bee sensible to me, as a player who understands what the human characters are saying, but it's not clear why the deadly creatures of the game would choose to do these things. Unlike in Metroid Prime, the game's goals are not organically integrated into its universe. Here, the fiction of the narrative seems to be fundamentally divorced from the intrinsic fiction of the game world.

This also comes into play with respect to combat. The tarantula and especially the scorpion fight with a few simple attacks and elaborate (motion-based) finishing moves reminiscent of God of War. While motion detection is not ideal for some of these, the core mechanics of combat are suitably engaging. The problem is that they are repetitive, and this is a problem not only because stinging a rat in the brain stops being interesting around the fifth time you do it, but also because the number and organization of the enemies conflicts with the game's intrinsic fiction. Combat is too frequent, your enemies attack in waves, and they never attack each other. There is no recognition here of the dangerous line predators tread with respect to energy expenditure, or the flight instinct that protects most prey. Even the weakest beetle is always up for a fight. Nor is there any consideration for the idea that territorial spiders are not going to attack as an organized body, and even if they managed it they would be at least as likely to attack each other as the nearby scorpion. No, the heroes of Deadly Creatures must fight their way through an army of opponents in exactly the same way as you could see in any standard 3D brawler. The encounters and enemy AI are informed by human sensibilities, right down to the heartbeat sound that warns you of low health.

The game posits that the deadliest creature is man, and so the scorpion, at least, must confront him. Of course, arachnids are actually rarely endangered by direct encounters with humans, because they mostly have the good instinct to hide. Deadly Creatures breezes past this reality, and discards essentially everything worthwhile in its combat system, so that we may end the game on a low note with a boss battle marred by nonsensical mechanics and out-of-place toilet humor. Even if I'd found it funny to sting a guy on the nuts one time, repetition would have drained the exercise of its joy.

If geography is a matter of scale, then our absurd dimensions must deny us many of the world's most amazing vistas. They exist at a size we cannot reach, in a world we can barely begin to experience. Deadly Creatures walks up to the edge of that vast uncharted space, gazes longingly upon it, and then spins on its heel and returns to the map of tried-and-true conventions. The game turns its back on the powerful fiction implied by its setting in order to embrace a story about humans, enemies that operate with human sensibilities, and objectives that make no sense for its protagonists. Deadly Creatures does not ask the player to be a scorpion or a tarantula, it asks the player to be a dude on a couch controlling a scorpion or a tarantula, and that is how a fantastic idea becomes a merely passable game.

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The falling action

Most games, even particularly interesting ones, are hamstrung by the fact that they end too soon. I don't mean that the gameplay itself ends too quickly; indeed most games would be served by chopping out a few hours. What I'm talking about here is that the story gets cut short. Whether RPGs, shooters, or brawlers, the finale of most games goes like this: climactic boss battle, short non-interactive cutscene, The End. Sometimes the short cutscene gets replaced with a long one, but the basic pattern is widely accepted. This hurts game narratives because it forces the player to assess the implications of the game's climax while almost entirely outside of the game world. It is left to the player to imagine what the world of the game is like after, and while this occasionally fits a narrative design it does not always do so. Fallout 3 reached a positively manic climax with Liberty Prime's assault on Project Purity, then dove right into an abrupt (and almost inexplicable) closing sequence. The recent downloadable content, "Broken Steel", feels like an attempt to add a real denouement to the Enclave arc, and in this it largely succeeds.

Most quests in "Broken Steel" differ from those of the main game in that they are essentially devoid of meaningful moral choice. The events of the add-on represent a single infiltrator's actions in a military campaign, and as such most of its quests amount to extended dungeon crawls. While a number of new and extremely powerful enemies appear across the Wasteland, none of them take the form of Enclave soldiers — the new Hellfire Trooper is not significantly more effective than his compatriots — and when this is combined with the elevated level cap, the effect is a slow reduction of tension throughout the adventure. Sure, things can get a bit hairy during some segments (particularly the harrowing encounter with Reavers in the Presidential Metro), but every time you face the Enclave they feel weaker than before. In the final act of the add-on, I infiltrated the Enclave's mobile base and single-handedly took down their elite Sigma squad in the space of a few seconds. Things never really got any harder after that.

Broken Steel doesn't have any grand finale to match that of the main story, but that's not the point. The new content isn't about a heroic charge against overwhelming odds to conquer a fortified position; it's about stomping bugs. There's no new antagonist to confront, nor even an old one, as the new segments don't seem to notice if you let Colonel Autumn live. All you have are progressively less dangerous firefights, gradually easing off the pressure until you demolish the Enclave's last stronghold with fire from the sky.

The accessory quests don't add much in the way of gameplay or new areas, but they do allow the player to consider the non-military impact of freeing Project Purity. Notably, the Wasteland still has problems — cleaning up the water didn't immediately eliminate the widespread contamination, mutation problems, or raiders. Solving some of those problems simply created new ones: now the challenge is to ensure that the distribution of the pure water goes smoothly. These quests don't really have much in the way of interesting moral choices, but they help put the player's earlier decisions in perspective, allowing him to reflect on the implications of the choices he made earlier.

The weakness of "Broken Steel" is that it has little to offer beyond violence. The quests are mostly straightforward battles, and allow none of the flexibility inherent to some of the main game's more interesting missions. The player can't side with the Enclave, or even use his powers of persuasion to help destroy his enemies from within. The only choice the player can make is to shoot or die, and while that approach has its uses it's unfortunate that players who take advantage of the full range of character customization available in the game aren't rewarded with interesting alternative routes to victory.

The impression one gets from interviews is that the developers at Bethesda believe players reacted negatively to the fact that their game ended. For me, at least, this wasn't the case at all. It wasn't that Fallout 3 ended, but how it ended that felt unsatisfying. The assault on the purifier didn't feel like a personal triumph, the conclusion of the adventure came as a surprise, and the in-universe reason for ending it was contrived and ridiculous. After the events of "Broken Steel", however, I feel like the game could have stopped without any trouble. The powerful weapons and increased level cap slowly deflate the tension of the narrative throughout this episode, and the final crawl through the Enclave's mobile platform feels like the player's expression of personal dominance over their military engine. Despite its weaknesses, this aspect of "Broken Steel" is quite welcome: it makes the player feel like the story really is at a conclusion, by moving the end of the game away from the climax of the story.

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Go for the Overkill

While many publishers have largely decided to approach the Wii by speeding up their casual-game assembly lines, Sega has started to position itself as a go-to source for more adult fare on the friendly little console. This is the strategy behind the upcoming first-person shooter The Conduit, as well as the already-released games MadWorld and House of the Dead: Overkill. MadWorld was blessed with a striking cel-shaded art style and gameplay that had the potential to use the Wii remote in really interesting ways. So it's perhaps surprising that Overkill, a light-gun game that holds the Guinness record for most swearing in a video game, turned out to be so much better. There is a phrase in the previous sentence that tells you why that happened.

Of course, spewing F-bombs in a game is not a guarantee that it will become a great experience, but that's not the point. Overkill's amazing proliferation of profanity is important as an indicator of what the developers were trying to do and how they succeeded. The entire design of the game is meant to evoke memories of the bloody, profane, and ridiculous B-movies of yesteryear. And it does, from the moment you see an ammo-draped stripper dancing behind the credits. The soundtrack, dialogue, and obvious continuity (and projection) errors are all reminiscent of the bad old movies, as are the posters that designate each of the playable chapters. Even the bosses serve as a sort of parade of monster archetypes from these flicks. Of course, this is all unbelievably crass, and you'll only like the game if you can find it at least as hilarious as it is revolting.

The game fits its target well because the on-rails light-gun game is a second-tier entertainment these days anyway, long surpassed by the greater freedom afforded in first person shooters. Overkill embraces this heritage, relying on its scoring system to enhance the replay value (although the "director's cut" expands the game also). The Wii's graphical limitations aside, the enemy models are noticeably ugly and low on polygons, and while this may not have been intentional it slots into the overall aesthetic anyway.

This isn't to say that Overkill doesn't have any brains (the zombies mutants need something to eat, after all). It's certainly aware of how crass it is, to the point where one of the characters (who previously unleashed a hilariously chauvinistic tirade against the game's female protagonist) offers a cursory feminist critique of the game in the closing scene. One could also read a little commentary on the history of the genre into the episode that takes place in a carnival. Light-gun games are, after all, the progeny of midway amusements, by way of the arcade, something you can also find in the carnival episode. Overkill keeps its commentary light, however, mainly aiming to evoke our memories of films we'd be embarrassed to admit having watched. The game knows that we know those movies were terrible in every way, and if it's trying to make a point beyond that, it isn't trying too hard.

MadWorld evokes memories of a particular movie: The Running Man. A whole city has been transformed into a bloody amusement called DeathWatch for... somebody, and the protagonist, Jack, must kill his way through the other competitors in order to stop the evildoers who arranged the whole mess. Like Overkill, MadWorld aims to get laughs out of the absurdity of its violence and an accompanying commentary track. MadWorld, unfortunately, never really gets going and even if it did it hasn't got a coherent destination.

The key problem with the gameplay is pacing. Overkill has a tremendous advantage in that its on-rails structure means that the developers can completely control the tension and the player's viewpoint at all times. This has its downside — once a player has "learned" a particular area it can get boring — but it also keeps the action going almost constantly. MadWorld, however, is a series of open areas in which the player can roam freely. This allows him to control his experience to some degree, but it also means that the player can feel somewhat directionless. Local shortages of enemies force the player to move, but in many levels enemies and interesting locations are clustered rather than evenly spaced. As a result, the player sometimes wanders aimlessly and can stumble into stretches where there just isn't enough to do. The scoring system exacerbates this problem by generally keeping players in these arenas for too long, allowing even its over-the-top violent antics to become repetitive and tiresome.

That explains how MadWorld falters as a game, but it also collapses as an idea. The cheesy visuals of Overkill contribute to its overall aesthetic intentions, and the black-white-and-red cel shading of MadWorld seems like it will do the same, targeting violent comics. But while Overkill dispenses with reality almost immediately and starts taking its story in more and more bizarre directions, MadWorld keeps itself firmly under the top, embracing lengthy cutscenes where people look shocked or frightened in freeze frame as various characters painstakingly explain the history of DeathWatch and the ordinary, workaday motives of the rich men who have organized it. Oh, they did it to make money, who could have guessed.

MadWorld doesn't even surpass its inspiration. Decades have passed, so now we have a lot more blood and dismemberment, but do we have a lightning-slinging opera singer wearing technicolor armor? MadWorld eschews incongruous beasts like Dynamo, instead giving us exactly what we expect from a game like this. The bosses always are and appear every bit as dangerous as Jack himself, and most of them are huge, violent monsters who can happily survive having an arm chainsawed off. It's surprisingly conventional, and even a little cowardly.

This extends to the concept of DeathWatch itself. The Running Man has a virtue in that it points the finger at its own audience: a totalitarian government organized the events, but the people watching, cheering, and wagering on the murderous game show were very clearly us. The rising popularity of MMA and the recalibration of football broadcasts as celebrations of the hard hit are evidence that the thirst for violent sport is as great now as ever. The target is there, and MadWorld's bloody excess and sportscaster-like commentators are like a loaded gun aimed right at it, so it is almost infuriating that the game never pulls the trigger. The Running Man at least afforded the possibility of an uncomfortable moment, when the viewer attempted to reject the idea that he was like the spectators in the film and then realized he'd just spent 90 minutes cheering on bloody murder too. MadWorld goes to some effort to assure its player that ordinary people are victims, only the jaded rich think murder is sport, something about pharmaceutical companies, blah blah blah. The reality Jack is every bit as brutal and disgusting as the thugs and aristocrats he's opposing, and he is us. You could do something powerful with that, if you were so inclined. MadWorld's developers apparently weren't.

House of the Dead: Overkill succeeds because it takes its every weakness and makes it serve the overall aesthetic. It's a relatively ugly game built on an unpopular mechanic that's linear by its very nature, and it works magnificently because of the B-movie horror context. MadWorld has every advantage over the other game when it comes to artistic style and gameplay mechanics, but like its directionless players it simply loses its way. It's not just that it doesn't commit; the game doesn't even seem to know what it could commit to. Overkill seemingly aims lower than MadWorld, but because it is built into a coherent aesthetic experience, it hits higher.

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cAMP gives CAP a twist

The catabolite activator protein (CAP), which plays a significant role in telling bacterial metabolism to digest sugars other than glucose, is a classic example of allosteric activation. Binding of the small signaling molecule cyclic AMP (cAMP) switches CAP into an active state that recruits RNA polymerase to certain metabolic genes. The biochemistry of cAMP activation is well understood, but the structural basis is not as clear, because a structure of the inactive protein was not available. This week in PNAS, researchers from Rutgers University and the University of Wisconsin-Madison report a structure of the free state of CAP that illuminates this allosteric effect.

Many excellent structural studies have examined CAP in its activated and DNA-bound form. CAP is a dimer, and each monomer has two domains: a DNA-binding domain (DBD) that recognizes its specific sequence, and a cAMP-binding domain (CBD). The monomers bind to each other through a coiled-coil interaction between two long helices. When CAP is activated it binds to DNA, with two helices (the recognition helices) sliding into the major groove and specifically identifying the sequences to which it should recruit the transcription apparatus. Without cAMP bound, CAP can still interact with DNA, but this interaction is of low affinity and not specific for any particular sequence. There are a number of ways this could conceivably happen, but it's difficult to be certain about any model in the absence of a structure of the free (apo-) protein.

In order to determine the structure, Popovych et al. used NMR. The 50 kDa size of the dimer means that it requires some extra effort for NMR work, but it is still well within the capabilities of the technique. The fact that the protein is a symmetric homodimer makes assigning the spectra somewhat easier, as the researcher only needs to deal with 209 residues rather than 418. The authors determined the structure using short-range distance restraints from nOe experiments, long-range restraints from paramagnetic relaxation enhancement, and angular restraints from residual dipolar couplings (RDCs). These angular restraints allowed the authors to unambiguously determine the relative orientation of the DBD and CBD in each monomer.

Getting that orientation right is key to the story here, as you can see from the image to the left. Here I'm showing you the DBD and coiled-coil helix (lower left) of a single monomer in the two different states. The activated CAP is in green (PDB code: 1G6N, and the apo- structure is in red (PDB code: 2WC2). You're looking down the coiled-coil, and the recognition helix is in a brighter color right at the front. If you superpose these structures on the bottom end of the coiled-coil, you can see that the recognition helix is rotated by 60° when cAMP binds. This twist of the DNA binding domain gives the recognition helices the right orientation and spacing to slide into the major groove of DNA and identify genes to activate. In the apo- state, these helices cannot both fit into the major groove simultaneously, explaining the low affinity and lack of specificity in that state.

Although the DBDs undergo a radical change in position following cAMP binding, they don't actually have any direct interactions with the signaling molecule, which binds down in the CBD near the coiled-coil helix. This helix, which links the CBD to the DBD, turns out to be key to communicating the allosteric signal. In the apo- state, the top part of this helix (near the DBD) is actually somewhat disordered and loop-like, not helical. Binding of cAMP to the CBD forms several contacts and causes several structural shifts that result in the formation of regular helical structure at the top of the coiled-coil. This in turn swings the DBDs around so that the recognition helices are in position to interact with target sequences (the authors provide a short movie of this in the Supplementary Information). A similar molecule, cGMP, that does not activate CAP, fails to make the key contacts with T127 and S128 that mediate this structural change.

The fact that the upper part of the coiled-coil is unstructured suggests that CAP may sample a range of conformations in the apo- state. This possibility is supported by one of the mutational studies in the paper. As you can see from Figure 5, a G141S mutation and binding of various effectors to the mutant causes the NMR resonances of DBD residues to shift on a line between the WT apo- and WT cAMP-bound states. This, in conjunction with the broadening of those intermediate peaks, suggests that the DBDs are exchanging between the two states on a timescale of microseconds. It seems quite likely that one or both DBDs in the inactive dimer occasionally samples the active conformation. In this model, the function of cAMP would be to stabilize, rather than enable the active conformation. The negative cooperativity of cAMP binding may help keep CAP switched "off" in the face of this conformational heterogeneity.

This study only dealt with a single protein, but the results are likely to be applicable to a number of systems. The allosteric mechanism described here seems to fit observations in at least some other members of the protein family to which CAP belongs. As such, this structural work and the dynamics investigations that will probably ensue are likely to provide important insights into a number of regulatory pathways in bacteria.

Popovych, N., Tzeng, S., Tonelli, M., Ebright, R., & Kalodimos, C. (2009). Structural basis for cAMP-mediated allosteric control of the catabolite activator protein Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0900595106

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Blog of the Round Table: Garden of Eden

In the April Blogs of the Round Table, Corvus asks us to design a game about a social issue we personally find troubling. There have been several stellar entries already, which you should check out from the main hall or using the pull-down menu at the end. Be aware that the subject matter of some of these games is fairly disturbing and may not be appropriate for all readers. My own design is rated E, because it is a light little planting sim I'll call Garden of Eden, which is a metaphor about sustainability and resource consumption.

The Design

In Garden of Eden, a player attempts to create the most aesthetically pleasing garden possible with a limited set of resources. The garden consists of several equally-sized patches of soil that the player chooses to seed with certain plants. Each plant requires a certain richness of dirt. The player can increase the richness of each patch's dirt by adding topsoil or fertilizer. By adding these elements the player can enrich the soil; every season of growth depletes the soil. The garden receives a score every season. So long as this score increases which each successive garden, or decreases by a small amount, the game continues. If the score falls by too much for 2-3 consecutive seasons the player is fired and the game ends. An online leaderboard built into the game interface shows high scores.

At the beginning of the game, the player has 8 patches under his control, and 16 different kinds of plant that he can grow (4 for each different season). The player has a warehouse containing a large but finite number of sacks of fertilizer and topsoil. A set number of these sacks (more topsoil than fertilizer) are deposited in hoppers on screen each season, and the player can move them from the hopper to a patch in order to enrich that patch of soil. Topsoil can only enrich the soil to a certain level, but fertilizer can enrich it infinitely. The only limit on soil richness, therefore, is the number of fertilizer bags in the hopper. The richness of each patch can be represented to the player either as a number or by the color of the soil.

Each plant requires a certain minimum soil richness to grow, and at this level will produce small flowers and a baseline score. Plants can be placed in soil that is too poor, but they will die and give no score. Making the soil richer than the minimum will produce a plant with more and larger flowers, and a much higher score. The return on investment is non-linear: enriching by X amount will increase the score by X^1.5 or more, up to the maximum available for a particular kind of plant. The minimum and maximum scores for each plant are known to the player, as is the amount of soil richness needed to achieve these scores.

Once the soil has been adjusted and the plants chosen for each patch, the plants grow to full bloom over the course of about 30 seconds. At this point the player can pause to admire his garden, or move to the next season. Higher-scoring gardens are always more interesting to look at — the plants themselves are larger, more detailed, and more beautiful. In addition, high-scoring gardens draw animals such as singing birds, deer, butterflies and bees, and human visitors who ooh and aah over the garden. Once a season is completed, the now-dead plants are removed into a rubbish bin. The player can choose to move them from there to a composting heap; after one additional season each plant left there will turn into one bag of topsoil. The intrinsic richness of the soil in each patch will decrease, and the cycle begins again.

With a high enough seasonal score or net score (added up across all seasons), new plants become available: these plants always have higher soil richness requirements, and also give higher scores with the same degree of non-linearity as soil enrichment. The player is shown when he will receive a new plant. The scores required are low initially so that new plants come rapidly, but as the game progresses it requires a higher and higher score to get a new plant. The player's highest-scoring season is also recorded on an online leaderboard, but it is not stored permanently. The score is only on the board as long as the player's game continues. The score is also removed temporarily after a few minutes (say, 10) of inactivity. The player sees the top ten, as well as his own position on the leaderboard and the 10 players who flank his score.

At a certain point, new patches become available, always delivered in groups of 16 — there are 5 extra groups of patches. Each time you receive one you get a one-time delivery of additional fertilizer and topsoil to the warehouse. In addition, because you now have more patches of soil overall, your hopper contains proportionally more fertilizer and topsoil sacks. This means that the maximum enrichment possible for a given patch of soil increases. It also means that more fertilizer and topsoil are removed from the warehouse each season. Something must be planted on every patch you have, and that plants on these new patches only count half as much towards your score as plants on your original patch. Additionally, the intrinsic soil richness of these patches depletes more quickly than the player's original 8 patches. The player cannot choose to refuse new patches.

Obviously the fertilizer will eventually run out, and as the intrinsic strength of the soil gets depleted the return from composting will not match the investment of topsoil needed to grow anything. If the player manages things properly he can quite easily keep the scoring decline slow enough that the game continues indefinitely. Once a section of soil is completely depleted and the player has no more topsoil, the game runs on its own. The soil in the six different groups of patches becomes cracked, dry, and barren as the seasons continue. The original set of 8 patches expands to take the shape of North America; the other groups of 16 patches, as they dry out, fill in a Mercator projection map of the earth. Once this happens the game ends; the player loses. If, however, the player manages to run the game for a very large number of turns, then he is told that he has won, and his best garden score from that game is permanently entered in the leaderboard. At no point prior to winning the game is the player told that this outcome is possible.

Aims of the Design

The game is meant to describe the problem (not a solution) of sustainability. In the metaphor fertilizer represents non-renewable resources and topsoil represents mostly-renewable resources. The composting process represents recycling efforts — the player must make a conscious mental effort to compost, and the recovery of resources is not totally efficient, in that many resources are lost and composting only returns the less powerful topsoil, not new fertilizer. The mechanics associated with the additional patches of soil represent a global drain of resources from poorer to richer nations, here represented by the currently-worst offender (the USA).

The mechanics of the game are meant to encourage the player to value a high-scoring garden with large, rich plants that suck down resources. This value is communicated to the player by the direct presentation (more beautiful garden, more visitors, more interesting sounds) and also socially, via the ever-present leaderboard. The requirement that the player actively interact with the game in order to keep himself on the leaderboard is meant to increase the volatility of the board, but also to keep each individual game moving forward. Additionally, ever-increasing growth or carefully controlled losses are valued by the game (by allowing progression).

Contemporary values romanticize the idea of a continuous march of progress, in which our success as a society is measured by how much bigger or better our things are compared to the things previous generations had. This is not just a view that informs our attitude towards technology, although it is most evident there; it also informed our latest real-estate disaster and the associated plague of suburban sprawl. The game is meant to communicate the idea that these values are self-defeating in a world of finite resources. Even if a small area can be sustained at a high level, this can only be achieved by taking resources from other areas at the cost of making their standard of life lower. Ideally I would tune the game so that it would be possible for a dedicated and careful player to run a modest garden indefinitely. To achieve this kind of success, or to "win" the game, the player must reject the "bigger, better" values implicit in the design. Because the game means to infect the player with these values, the upshot is to make the player question himself.

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Who's in the party?

Along with the Vintage Game Club, I have recently been playing Chrono Trigger, a game widely hailed as one of the best console RPGs ever made. While the game has many attributes worth praising (not least the continuity of world and battle screens), I was particularly interested by its combination attacks. Almost any two characters that are in the combat party can combine their unique individual battle techniques into more powerful "Dual Techs". For instance, Lucca can charge Crono's sword with fire while he performs his "Cyclone" attack to create the "Fire Whirl" tech. Frog and Ayla can combine their weak individual healing skills into the "Slurp Kiss" that slightly heals the whole party. I think it odd that this approach has seen so little use subsequently, since it seems like modulating abilities based on party composition has great potential for infusing characterization into the combat mechanics.

Of course, most role-playing games, especially in the JRPG genre, give the player characters with complementary abilities or specialized roles, so that certain rosters are somewhat more effective than others. Having the characters explicitly combine their attacks is much less common, and is typically reserved for high-powered finishing moves, as in Kingdom Hearts II or Tales of Symphonia. Chrono Trigger differs from these games in that its combination techniques span a spectrum of opportunity costs (also, the animations are short). Some of them do require a significant investment of MP and can only be used sparingly, but others have a low enough cost that they can be used almost every turn. As a result, the shared abilities are almost as important as the individual abilities to the tactical evaluation of a particular party composition.

This had an interesting effect later in the game, after I managed to get Magus to join my party. Magus is a strong and versatile character with hard-hitting magic attacks of several different elements. Considering just his individual abilities he's a good character to have in the party. Yet, I almost never used him because he didn't have any dual techs at all. He couldn't increase his effectiveness by teaming up with anyone, and rather than give up the tactical flexibility, I left him out. It felt mechanically like he just didn't fit in with the party, an appropriate note because he is a major antagonist for much of the game. The mechanics of the game isolate him in battle just as the story suggests he would be isolated in the group.

This seems to me like the sort of thing that should be done more often. The enormous cast of many JRPGs means that most of the playable characters and their relationships never really get fleshed out, despite the hours of cutscenes. Why not use the combat system to do some of this work? Envision a set of party dynamics, and use the availability of combination attacks to illustrate those dynamics. If you imagine that Marle is jealous of Ayla and Lucca, creating friction in the group, express this by limiting their combos or making them less powerful. Then, when the player needs Lucca in the party for her fire attacks, the tactical limitations would encourage the player to respond by removing Marle from the combat group. The player's choice of party members remains an act of play, but through the manipulation of the mechanics it becomes an act of narrative characterization as well.

We can push this further by making the available combos respond dynamically to narrative. Events in the story that bring two characters closer could make new combination attacks available to them. When the relationship between two characters changes, the set of shared techniques could be adjusted to reflect the difference. This need not be tied to the planned story: a relationship system like that of Tales of Symphonia could be used to guide the evolution of combination techniques.

I've seen a couple of excellent essays (from Jay Barnson and Nayan Ramachandran) recently trying to dissect what's wrong with the JRPG and suggest ways that the genre could be revitalized. My own advice is to let the gameplay tell the story, too. Persona 4 and The World Ends With You both did this, in different ways, but you can still apply the lesson even if you aren't so unconventional. Use the gameplay of party dynamics to reinforce the story of personal relationships. Make "these characters belong together" part of the player's thinking in every phase of the game, and the stories of those characters may gain more resonance.

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Drugs disrupt DHFR dynamics

One of the most-studied cases of the relationship between dynamics and catalysis is the bacterial dihydrofolate reductase (DHFR). DHFR catalyzes the reduction of dihydrofolate to tetrahydrofolate while oxidizing the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). As part of this catalytic process, a region of the protein called the "Met 20 loop" switches from a "closed" state that shields the active site from solvent to an "occluded" state that separates the substrate from the cofactor. NMR studies of DHFR structural dynamics have correlated the protein motions with the chemical changes. In a recent study appearing in Structure, researchers from the University of North Carolina show that the binding of inhibitors such as methotrexate (MTX) and trimethoprim (TMP) appears to uniquely disrupt the dynamic networks of DHFR.

Previously, seminal work from the lab of Peter Wright surveyed the dynamics of DHFR in every step of its reaction pathway. Boehr et al. determined that structural fluctuations in each complex represented motions towards the next step in the reaction. The conformational exchange rates they obtained from their relaxation-dispersion experiments closely resembled the rate constants that had been independently determined for the chemical steps. In almost every complex the conformational exchange was widespread, affecting residues in both the substrate and cofactor binding sites, as well as important distal locations such as the Met 20 loop.

Because the existing work from the Wright lab hewed as close to the natural substrates and products as possible, Mauldin et al. chose to examine the dynamic effects of inhibitor binding to DHFR. Like Wright's group, they used relaxation-dispersion experiments to identify conformational changes taking place on the μs-ms timescale. In the NADPH:DHFR complex the motions are widespread, encompassing the substrate binding site, the Met 20 loop, and distal locations. Binding of either inhibitor eliminates about half of this dynamic network and dramatically reduces the fluctuation rates of those residues for which conformational exchange continues to occur.

Based on their fits of the exchange rates, Mauldin et al. conclude that the substrate binding pocket moves in a way that mimics the enzyme's normal motions in the transition from its closed state to its occluded state. The long-range conformational changes that actually complete this transition, however, have been completely quenched. With the inhibitors bound, DHFR is like a car that's turning over but won't start. Part of the enzyme is still moving in exactly the right way to proceed along the reaction coordinate, but for some reason this motion doesn't catch on throughout the protein.

In order to gain a more complete understanding of the dynamic effects, Mauldin et al. performed experiments to identify the motion of the protein on the ps-ns timescale. Analyzing the dynamics of methyl and amide resonances using the Lipari-Szabo model-free formalism, the authors realized that inhibitor binding did cause long-range changes in dynamics, just in a faster regime. Where the natural substrate complexes have motions that occur hundreds or thousands of times per second, the inhibitor-bound forms have (smaller) motions that occur millions of times per second. Because these altered motions encompass the Met 20 loop and surrounding residues, the authors argue that they reflect abortive attempts by the protein to transition into the occluded state.

Although these inhibitors do not appear to change the protein's overall conformation, they produce long-range dynamic effects on short timescales and quench distal motions on intermediate timescales. The binding pocket appears to still be experiencing fluctuations related to the transition between the closed and occluded conformational states, but the mechanism that couples the binding site dynamics to the motion of the loop that defines these two states appears to be broken.

The million-dollar question is this: do drugs alter DHFR dynamics because they inhibit the chemistry, or do these drugs inhibit the chemistry because they alter DHFR dynamics? Quenching dynamics costs energy in the form of conformational entropy, and it may be possible to tune a drug for improved efficiency by blocking the binding site without altering the dynamics. This is only true, however, if the dynamics don't matter to successful inhibition. On the other hand, if blocking the conformational switching of the Met 20 loop inhibits the enzyme, then drugs can be designed for that angle of attack as well. In the case of a protein like DHFR, where the bacterial enzyme has similar activity but a very different structure from its human equivalent, drugs that target regions other than the active site may significantly reduce side-effects. As a result, protein targets that were previously off-limits due to shared chemistry may become tractable due to divergent dynamics and structure.

Mauldin, R., Carroll, M., & Lee, A. (2009). Dynamic Dysfunction in Dihydrofolate Reductase Results from Antifolate Drug Binding: Modulation of Dynamics within a Structural State Structure, 17 (3), 386-394 DOI: 10.1016/j.str.2009.01.005

Boehr, D., McElheny, D., Dyson, H., & Wright, P. (2006). The Dynamic Energy Landscape of Dihydrofolate Reductase Catalysis Science, 313 (5793), 1638-1642 DOI: 10.1126/science.1130258

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Critical Thinking Compilation: BioShock

BioShock is the rare game that really does change the way we think about video games, if for no other reason than that it has turned up as an example in almost every discussion of game style, mechanics, story, or design that has been written since its initial release in 2007. BioShock has received excessive adulation, a much-discussed backlash, and even a backlash to its backlash. Discussions of the game spawned the most popular jargon in games writing. So much has been written about BioShock that I could only put my hands on a fraction of the material without driving myself nutty as a splicer. Thus, this constitutes only the first draft of a survey of critical thinking on a game that will likely be regarded as a classic.

"Is a man not entitled to the sweat of his brow?"

One of BioShock's most compelling features is that it details an interesting philosophical system and then uses it to frame an ethical question. The underwater utopia of Rapture was founded by an industrialist named Andrew Ryan on a system of principles much akin to Randian Objectivism, so much so that John Lanchester argues in the London Review of Books that BioShock is the only popular work in recent years to give Rand a drubbing. Lorenzo Wang fleshes out this case in his rich and interesting essay "BioShock Explained". In his view, the game attacks two key flaws of Rand's philosophy: society can never sustain its ideal state (somebody must, after all, scrub the toilets), and free will, even in the land of plasmids, is limited. Jay Barnson felt that the game critiqued the intrinsic short-sightedness of the market, which is regarded as all-wise by the lovers of laissez-faire economics.

This interpretation of the game's attitude towards Objectivism was not universal, however. Shamus Young interviewed an Objectivist on the subject, who argued that BioShock really aims its criticisms at the idea of philosophical certainty. In his very interesting Marxist critique, Richard Terrell lays out a case that the game conditioned the player to accept the principles of Rapture's economy. In his view, the choice to attack the Big Daddies (and possibly the little sisters) makes the player part of the oppressive capitalist regime. Justin Keverne argued that the mechanics of the game suggest that one's goal is to acquire power in order to gain the ability to acquire further power.

At this point, designer Clint Hocking felt that BioShock went off the rails in a certain sense. In his essential essay "Ludonarrative Dissonance in BioShock", Hocking argues that the game presents the player with two conflicting contracts. The gameplay establishes that the player must serve his own interest in order to advance, while the story forces the player to serve others in order to advance. Our attempts to deal with or ignore this tension are them mocked by the game's central twist.

"How can you do this thing? To a child?"

The philosophy of rational self-interest provides context for the story, and also for a moral choice that the player makes. When Rapture's lumbering Big Daddies are defeated, the player may choose the fate of "little sisters" they protect. If he rescues a little sister, he receives a small amount of ADAM that he can use to purchase plasmids, tonics, and other upgrades, and the girl survives. If he chooses to "harvest" the girl, he gets a great deal more ADAM, but she does not survive. For Leigh Alexander, saving the girls is like saving Rapture's last bit of innocence. In her view, the whole saga with ADAM was like a child's wish, and saving the girls is a way of forgiving Rapture for making that wish, despite the destruction it caused. Yet many did not find this choice to be compelling. As Wes Erdelack notes, in BioShock as in many other video games, the moral choices are too simplistic and do not feature a sufficient challenge to "goodness".

One frequent complaint about the rescue / harvest choice is that if the player rescues enough little sisters, then the twisted Dr. Tenenbaum will leave a Teddy bear at a nearby vending machine, stuffed with plasmids, ammo, and extra ADAM she has presumably harvested on her own. In a thorough critique of this moral choice, D. Riley argues that the benefits of rescuing are sufficiently great that the whole system is neutered. Duncan Fyfe concurs, and wonders how the developers could have gotten this so wrong.

This opinion is not universal. Leigh Alexander believes that approaching the rescue / harvest choice with a cost-benefit analysis is too limiting. In her view the question to ask is not what you want to get but who you want to be. As she says, "The merit of choice in games may not be what we get from it, but when done this richly, how it feels." Bonnie Ruberg feels that the gameplay is really there to expose the selfishness of gameplay tactics in general. I also felt that the near-equivalence (in economic terms) of the two choices was making a point about the hidden values of games.

Some also argue that the harvest choice is not compelling because the player is spared having to watch the act or even see the body that remains. D. Riley's previously noted essay includes a comment on this point, and I make a similar case in my own essay, "Ecce, soror". Nels Anderson acknowledges that this may be so, but points out that on-screen child murder by the protagonist of a game simply could never get by ratings boards or the easily-outraged public. Nonetheless, he argues that the nursery scene provides some of the necessary emotional impact.

And perhaps the little sisters aren't even the real focus of the moral choice. Justin Keverne argues that the player's real moral choice is whether to attack the enslaved Big Daddies for his own personal gain. Unlike the other denizens of Rapture, the extremely dangerous Big Daddies won't harm you unless you attack them first. For Glenn Turner, the choice to put down a Big Daddy was harrowing because of the little sisters' reaction. Gene Koo felt that becoming one of these lumbering behemoths brought the game's emotional and philosophical threads close to each other. In his view, however, this didn't quite succeed, because the player has no choice about whether he becomes a big daddy.

Whether the rescue / harvest choice was compelling or not, BioShock at least invited a fresh consideration of the meaning of moral agency in games, according to Leigh Alexander. She also pondered whether our behavior in the game would change if our peers were aware of it (perhaps through achievements or trophies).

"A man chooses; a slave obeys."

Choice is the subject of BioShock's most compelling moment, the confrontation with Andrew Ryan. This moment twists the preceding exposition of the ideas of rational self-interest into a commentary on the nature of gaming itself. Wes Erdelack views BioShock as parable about gaming, highlighting the fact that the feeling of agency is always an illusion. The Graduate School Gamer notes in an essay comparing BioShock to Braid that "The player can only converse with the text within the confines of the game's design and always remains at the will of the designer." The illusion of choice is not a subject unique to gaming, according to Roger Travis. He notes that the non-choice of killing Ryan resembles Achilles' non-choice to join battle in the Iliad.

The first-person perspective heightened the impact of the climax. Matthew Gallant regards the first-person viewpoint as essential, especially in moments like the confrontation with Ryan. The personal choices at the center of the game couldn't be the same if the player's eye into the game world didn't seem to make him a part of it. Sinan Kubba shares Gallant's skepticism about a BioShock movie, feeling that the immediacy and immersion of the game's perspective could never truly be replicated in film. The praise was not universal, however: Brad Gallaway felt that the silent, first-person protagonist interfered with the narrative at several important points. He especially felt that the internal logic of the game collapsed when the player injected himself with a plasmid for the first time.

The more widely-expressed complaint about the plot, and specifically the encounter with Ryan, is that there is far too much game after it. The denouement of the game is widely recognized as its weakest segment, a fact that Josh Birk explains by pointing out that BioShock, like many other games, has more backstory than story. The confrontation with Ryan is the culmination of the fascinating backstory, leaving the rest of the game to become little more than the tale of a man with a gun out for revenge. Moreover, the game doesn't exactly free the player up to make his own choices after its climax. The player continues to obey a character, only now it is Tenenbaum rather than Fontaine. BioShock refuses to engage this dilemma, which is a significant source of frustration for Duncan Fyfe. Chris Dahlen also found this troubling, because the game's most compelling character (Ryan) has so much agency and the player has so little. As he puts it, "...within the game, you never become a man. The only choice you have is to stop playing."

Aside from Ryan, surprisingly few writers have gone in depth on the characters of the game. A worthy exception to this rule is Leigh Alexander's examination of the bizarre Sander Cohen. She relates his personality to people she knows from her theater background, seeing in him a metaphor for the whole backstory of the game. In her view, Cohen is "a brilliant character not only for his spot-on characterization, but for the way his endless wrestling with 'the muse' is a perfect metaphor for the consumptive nature of Rapture in general."

"I know why it has to be children, but why just girls?"

Without focusing a spotlight on particular characters, several interesting pieces have examined the role of women and femininity in BioShock. Although creepy little girls are a staple of the horror genre, as Leigh Alexander has noted, BioShock uniquely gives the player power over their fate. Bonnie Ruberg found the female enemies in the game particularly disturbing, and wondered whether their horrific power drew from the simple fact of their gender. She also found the power relationship between the player and the little sisters to be troubling, and agreed with a Penny Arcade comic suggesting that it had overtones of pedophilia.

Nels Anderson indicates that the design of the girls is intended to evoke sympathetic feelings, but what attitude does this imply on the part of the player and developers? In a comprehensive critique, Richard Terrell argues that BioShock pervasively trades in patriarchal values because it "depicts women as weak, emotional, submissive, and nurturing and men as strong, and protective...". The little sisters are portrayed as helpless human commodities, and for much of the story Diane McClintock equates her self-worth with physical attractiveness. Moreover, Dr. Tenenbaum's redemption comes through an acquiescence to patriarchal ideas of motherhood. Terrell's analysis encompasses the mechanics of interacting with female characters as well.

"I chose the impossible. I chose... Rapture"

It's easy to understand how Cohen, Tenenbaum, and so many others could have chosen Rapture. For Ed Borden, the environment was key to BioShock's immersion of the player. The crumbling city arrests the player's attention and inspires his curiosity. Glenn Turner felt the same way, arguing that the art design was perhaps the game's best feature. For Richard Naik also, the selling point of the game was Rapture's auditory and visual design, overwhelming all of the game's shortcomings. The art design unified the disparate levels, making the world of Rapture feel like a coherent whole and maximizing the emotional impact on the player, as Tom Cross explains in "Surviving Rapture".

Part of the power of the environment was the way in which it was used to tell a story. Steven O'Dell compared the player's journey through Rapture to a guided tour of a dying city, one in which the enormously detailed spaces tell a story through the way they are designed. Wes Erdelack points out that the much-loved environmental storytelling of Fallout 3 has some roots in the construction of Rapture's spaces. While the audio logs constitute the most powerful storytelling in BioShock, the game's spaces allow the player to play detective and reconstruct his own history for the game world. Careful construction of spaces and traversals played a significant role in player experiences as well, as Simon Cooke explains. Every time the player encounters a massive set piece, the developers use clever design to make sure he is able to see every bit of it.

"...bugger gets into his 'ead that he's gonna go down guns blazing."

Also making sure that the player can see every inch of the city is the fact that he simply can't die in it. In some respects, the Vita-Chambers that resurrect the player after every fatal encounter resemble a streamlined checkpoint system (much like Elika in the later Prince of Persia), and Scott Juster places them among a number of ways that BioShock used narrative elements to disguise common gameplay tropes. Nonetheless, the Vita-Chambers were widely criticized. This goes beyond the hardcore player's lament, articulated for us by Josh Bycer, that resurrection makes the game 'too easy'. Josh Birk complains that they interfere with the scare factor in the game and the logic of weapon collection. Justin Keverne points out that they encourage the player to take the path of least resistance, though for him the draw of using the plasmid powers was enough to keep him playing fair. Not so for Richard Terrell, who felt that the chambers too strongly encouraged the wrench / revive / repeat approach to combat, and demoted the central activity of the game, which he felt to be shooting. Moreover, he argues that the effective immortality of the player weakens the psychological impact of the game in his psychoanalytic evaluation.

Another way to conceive of the Vita-Chambers might be as a resurrection spell, an apt comparison since BioShock had so many RPG elements. In fact, Richard Terrell felt the game's mechanics tended more towards the role-playing side, in particular because the almost nonexistent cover system forced the player to behave like a bullet sponge. He compares tactics in BioShock to the attack / attack / heal approach common in RPGs. Justin Keverne comments on the oddity of this, as the Vita-Chambers actually make healing and health packs totally superfluous in all but the final battle. Writing for Eludamos, Matthew Weise connects BioShock with RPG roots originating in Ultima Underworld.

BioShock resembled RPGs in even less flattering ways as well, specifically because of its fetch quests. These quests added numerous objectives that muffled the story, in Duncan Fyfe's view. Rather than engaging in a breakneck pursuit of Ryan (or Fontaine), the player spends his time mucking about Rapture looking for 7 vials of bee spit. This quest temporarily scared Tom Armitage right out of the game, even though it ultimately led to some interesting exploration. He warns, "I was thrown by the instructions the game gave me..." saying that even a good game can be derailed by players' bad memories of similar quests.

And despite the "too easy" vita chambers and exciting plasmid powers, the conventions that BioShock embraces limit its audience. Although Lanchester praised BioShock's consideration of Randian philosophy, he criticized the game because even its modest difficulty would keep it from being experienced by a broader culture that might genuinely appreciate it.

Author's Note

If you go to any of these blogs and search them for the word "BioShock" you will come up with dozens of posts. The game has become a kind of yardstick by which we measure others, and a rich source of examples to illustrate points. I attempted to limit what I included here by stipulating that the post must be at least 50% about BioShock, and that it should be in fully developed paragraphs rather than bullet points. I'm not sure I actually ended up holding to that, but that was at least the approach I tried to apply. I am positive I left some excellent posts out, mostly by failing to find them in the vast wilds of the internet. If I omitted your dissertation on the semiotics of dentistry in the context of Rapture, it is probably because I didn't run into it while I was doing my survey. Would you kindly let me know about it in the comments, via twitter, or with an e-mail?

Last updated: 4/6/09

Future updates of this compilation have moved to Critical Distance. Look for this and other great critical compilations (by myself and others) there.

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You can't put a price on your soul

BioShock's most famous moral choice concerns the fate of young girls who are wandering through the undersea city of Rapture. The player gets to decide whether these girls live or die, and in a game that features a civilization built on the principle of laissez-faire one expects economics to play some kind of role in this decision. Many writers, however, have complained that it does not, and that the choice has too little of an effect on one's ultimate experience of the game. Leigh Alexander and Bonnie Ruberg, among others, have suggested that something more sophisticated is going on here, and in other aspects of the player's behavior in Rapture. The difficulty of resolving the rescue / harvest choice in BioShock by a cost-benefit analysis illuminates the little-discussed value of selfishness embodied by most gameplay.

Ludonarrative dissonance and the player's choice

Clint Hocking's famous critique of BioShock accused it of developing a case of "ludonarrative dissonance", or tension between the message of the mechanics and the message of the story. As he put it, the mechanics offer a "ludic contract" that tells the player to seek out power (in the form of weapons, ammunition, plasmids, and ADAM) in order to advance in the game. This contract encourages the player to adopt a philosophy of rational self-interest much like that espoused by Andrew Ryan, the founder of the game's undersea dystopia. The story, by contrast, offers a "narrative contract" that tells the player to help others (initially Atlas, later Tenenbaum) in order to advance. In Hocking's view, the player can choose to accept or reject the ludic contract by harvesting the little sisters (maximizing his take of ADAM) or rescuing them, respectively. However, because the overall narrative of the game is fixed, the player cannot really choose to accept the philosophy of rational self-interest, and the climactic confrontation with Ryan seems to ridicule the player for putting up with that limitation.

Subsequent discussions of the game took a curious turn, however, in that aspects of the rescue / harvest choice itself were accused of being the centerpiece of dissonance. Wes Erdelack's excellent discussion of ludonarrative dissonance is a case in point, although I believe this take on the matter originates with Jon Blow. The essence of the complaint in this case is that the story sets up the player's choice to save or kill the girls as crucial, the consequences of the choice amount to almost nothing in terms of the gameplay. Although the player obtains significantly less ADAM when he chooses not to harvest the little sisters, repeatedly rescuing the girls causes Tenenbaum to give him gifts containing additional ADAM, ammunition, tonics, and a unique plasmid. As a result, in terms of strict gameplay consequences, the choice the player makes with respect to the fate of the little sisters doesn't matter. In essence, the game doesn't allow the player to make a choice about the philosophy of rational self-interest because both options are compatible with that philosophy.

These discussions all assume that dissonance ought be avoided, but as Michael Abbott has noted, it has its uses. The odd discordant note may offend the ear, but it draws attention and forces the observer to consider the artwork more carefully. The seeming mechanical equivalence of choice may be the conceptual error of a foolish team, or an intentional effort on the part of the developers to make the player consider the conflict presented more carefully. If we interpret this dissonance in BioShock as signal rather than noise, BioShock indicts the value systems of games and gamers.

You are only a link in the great chain

The philosophy of rational self-interest lies at the heart of both kinds of dissonance in the game. Andrew Ryan intended Rapture to be a utopia with no governing principle other than man's own self-interest, and regards those who seek the protection of a government to be little more than parasites sucking society dry. He builds Rapture for himself. His primary antagonist, Frank Fontaine, embraces this philosophy in a more nuanced way. He too only acts in his rational self-interest, but rather than Ryan's reactionary hatred of charity, Fontaine sees the feeding and housing of the poor as a transaction in which he purchases loyalty. They are very different men — Ryan wishes to build a society, Fontaine aims only to plunder one — but their heuristics for moral decision-making are identical. Whether either of them fairly depicts Randian Objectivism is an interesting but ultimately pointless debate; their self-interested philosophy most resembles the implicit morality of video games.

Obviously I don't mean to attribute a moral system to games like Guitar Hero, but most games that feature a player-controlled avatar operate on a moral system much like Fontaine's, in which all the world is the player's for the taking. Certainly most games try to cover this with a veneer of a story that somehow involves saving the world, but this motivation rarely takes center stage in the player's mind. In almost every game in existence, the player is encouraged to kill everything he encounters and plunder every chest he sees in order to win the game. Role-playing games often acknowledge this, usually through some abstracted morality system as in the Fable games. Yet in these cases only the extent of the behavior is criticized, not the fundamental attitude. So long as a chest or trove lacks any obvious owner, the player will not be criticized for taking it. The primary value of these games is always selfish advancement motivated by hedonism.

"Never play a man for the short con when you can play 'em for the long one..."

In keeping with this value set, we expect our options in games to be clearly differentiated in terms of their utility with respect to our self-interest. Because this is not operationally true of the rescue / harvest choice, many players were frustrated or confused. This statement from Duncan Fyfe, is typical of the protests:

It's hard to understand why this is the case because it's such a fundamentally simple and classic philosophical debate. You can make the rational, self-interested choice to gain as much resources as you can from this one interaction. Or you can sacrifice/minimise your short-term reward in favour of long-term benefit. This is how it's presented to you and it's basic political philosophy. It's realism versus idealism. You'll get less Adam for rescuing the little sisters but there's the promise of a greater reward down the line.

The problem with objections like this is that delaying gratification doesn't put philosophical ideas into opposition at all. The debate over long-term benefit vs. short-term benefit is just an argument over what constitutes self-interest, or what is the more "rational" route towards actuating it. Reducing the rescue / harvest choice to long-term vs. short-term gains merely recapitulates the contrast between Ryan's short-sighted anti-altruism and Fontaine's "long con", but neither of those options entails a rejection of rational self-interest.

It's not the choice you make, it's why you make it

If you want the player to really consider the implications of the philosophy of rational self-interest in the rescue / harvest choice, there are only two real ways to do it. The first is to make the rescue choice truly oppositional to the player's expected self-interest, i.e. give him no ADAM or very little for taking this route. The obvious disadvantage of this approach is that the game now becomes highly skewed. Although the vita-chambers relieve difficulty in the typical sense of dying and thus being unable to continue, the game will certainly become a much more frustrating and challenging experience. A gamer who played through such an experience might come away with the impression that the game was designed to convey the idea that Ryan and Fontaine were right, that he should have chosen killing.

The other alternative is to arrange gameplay in such a way that "rational self-interest" no longer constitutes a reasonable standard for decision, and this is what the choice looks like. The point is not to give the player a way to reject the philosophy in the decision, but rather to draw his attention to the fact that this was such a large part of his decision-making all along. Rational self-interest cannot tell us what to do with the little sisters because both options fit this mindset. Thus, the arbiter of judgment is your conscience, as it is in real life. The fact that costs and benefits of the options you can take with the little sisters don't conform to our expectations about "moral choice" in a game invites us to examine the values that inform those expectations, and the ways in which games express or exploit them.

Games have implicit values — do as you're told, take what you will — that we might disagree with were we forced to acknowledge them explicitly. Because these values are both hidden and integral, it is difficult to force the player to confront them without a shock or a feeling of confusion. The dissonance in the rescue / harvest choice may be intended to inspire the latter. If so, then both the confrontation with Ryan and the choice for the little sisters are commentaries on the nature of video games. Despite the ever-increasing promises of freedom, games have always played you the same way Fontaine played Jack. And regardless of how you feel about Ryan and Fontaine, you have always played games as if you were them. BioShock elaborately displays the callousness and evil of these men, and through them, you. How do you feel about that?

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